Pharmaceutical composition comprising adenosine derivative for prevention and treatment of retinal disease or optic nerve disease

ABSTRACT

The present invention relates to a pharmaceutical composition, an oral administration agent and an eye drop for preventing or treating retinal diseases or optic nerve diseases comprising the compound represented by the Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient and the pharmaceutical composition, the oral administration agent and the eye drop can effectively prevent or treat retinal disease or optic nerve disease.

TECHNICAL FIELD

The present invention relates to a composition for preventing ortreating retinal disease or optic nerve disease comprising an adenosinederivative.

BACKGROUND ART

Adenosine is a substance that performs many physiological functionsthrough a receptor on a special cell membrane and adenosine that existsoutside the cell acts as a neurotransmitter in many physiologicalsystems, and generally it compensates for the hyperactivity of a givenorgan and acts to protect against the harmful effects of stress(Jacobson, K A et al., J. Med. Chem., 35, 407-422, 1992). This action isdue to a partially generated negative feedback loop, which attempts toreduce the energy demands of the cells by adenosine produced by thebreakdown of intracellular or extracellular ATP (adenosine triphosphate)and increase the supply of oxygen. Adenosine is important formaintaining homeostasis of essential organs such as the brain, heart,kidneys and for example, the administration of adenosine agonists fromthe outside to the brain has been shown to have neuroprotective effectsand it is also known to be involved in pain, cognition, exercise orsleep.

Adenosine receptors have been classified into P1 and P2 receptors,respectively, through pharmacological studies and molecular cloning todate. Adenosine acts as a substrate for the P1 receptor, and ATP, ADP,UTP and UDP act as a substrate for the P2 receptor, thereby expressingphysiological activity. Among them, four different subtypes of adenosinereceptors were identified as P1 receptors, which are classified as A₁,A₂ or A₃ according to affinity for ligands, distribution in the body,route of action and the like, and again A₂ is classified as A_(2A) andA_(2B). These adenosine receptors are a class of the G-protein-coupledreceptor group, and adenosine A₁, A_(2A) and A_(2B) receptors have beenpharmacologically identified using many selective ligands, but theadenosine A₃ receptor was first discovered in 1992 (Zhou, Q. Y, et al.,Proc. Natl. Acad. Sci., USA, 89, 7432-7436, 1992) and much research hasbeen performed to confirm the pathophysiological function of thisreceptor.

Adenosine A₁ and A₂ receptor agonists are mainly derivatives ofadenosine, which have been actively studied as antihypertensive agents,antipsychotics, arrhythmia drugs, fat metabolism inhibitors (diabetesdrugs) and brain protectors, and their antagonists are xanthinederivatives or a fused bicyclic ring, and is being developed as anasthma therapeutic agent, antidepressant, arrhythmia therapeutic agent,kidney protectant, Parkinson's disease therapeutic agent andintelligence development agent, etc. Nonetheless, what is currentlycommercialized is only adenosine itself, which is used for the treatmentof supraventricular tachycardia, and dipyridamole, an adenosinetransport inhibitor that is used as an adjuvant for warfarin to preventblood clotting after heart surgery. The reason why suchcommercialization is not smooth is that because adenosine receptors arespread all over the body, it is due to various pharmacological actionsaccompanied by the activation of the receptors, and that is, there is nocompound capable of activating only the adenosine receptors of a desiredtissue.

Among the adenosine receptors, the adenosine A₃ receptor is the mostrecently discovered receptor unlike the well-known adenosine A₁ and A₂receptors, and its role is not well known and many studies are ongoingto develop selective receptor modulators. To pharmacologically study theadenosine A₃ receptor, three radiolabeled ligands are used, which are[¹²⁵I]ABA(N⁶-(4-amino-3-[¹²⁵I]iodobenzyl)-adenosine,N⁶-(4-amino-3-[¹²⁵I]iodobenzyl)-adenosine),[¹²⁵I]APNEA(N⁶-2-(4-amino-3-[¹²⁵I]iodophenyl)-ethyladenosine,N⁶-2-(4-amino-3-[¹²⁵I]iodophenyl)-ethyladenosine) or[¹²⁵I]AB-MECA((N⁶-(4-amino-3-[¹²⁵I]iodobenzyl)-adenosine-5′-N-methylcarboxamide,N⁶-(4-amino-3-[¹²⁵I]iodobenzyl)-adenosine-5′-N-methylcarboxamide).Through pharmacological studies using the radiolabeled ligand, it isdemonstrated that when the adenosine A₃ receptor is expressed in ChineseHamster Ovary (CHO) cells, the A₃ receptor has an inhibitory action ofadenylyl cyclase, an enzyme that produces cAMP from ATP, and when the A₃receptor is activated by an agonist, GTP-dependent phospholipase(Guanosine triphosphate-dependent phospholipase C), an enzyme thatbreaks down phosphatidyl inositol in the brain to produce inositolphosphate and DAG, was activated (Ramkumar, V. et al., J. Biol. Chem.,268, 168871-168890, 1993; Abbracchio, M P et al., Mol Pharmacol., 48,1038-1045, 1995). This discovery makes it possible to explain thepossibility of a response pathway by A₃ receptor activation in brainischemia and it is because this secondary transmitter system means aresponse pathway of neurological injury in brain ischemia. In addition,it is known that agonists of the A₃ receptor inhibit the release oftumor necrosis factor (TNF-α), an inflammatory mediator, and alsosuppresses the production of inflammatory mediators, MIP-1α,interleukin-12 and interferon-γ and protects the heart as well as theprotective effect against brain diseases such as epilepsy. Inactivationof the adenosine A₃ receptor causes the release of inflammatory factorssuch as histamine from mast cells, acts to contract the bronchi, andalso causes apoptosis in immune cells. Therefore, since adenosine A₃receptor antagonists have potential for development as anti-inflammatoryand asthma therapeutic agents, it is possible to develop new therapeuticdrugs for various diseases such as asthma, inflammation, brain ischemia,heart disease and cancer, if compounds with pharmacological selectivitycan be developed.

Among the materials that have been researched and developed to date, arepresentative human adenosine A₃ receptor agonist is nucleoside family,N⁶-(3-iodobenzyl)-5′-(N-methylcarbamoyl)-adenosine (IB-MECA) andN⁶-(3-iodobenzyl)-2-chloro-5′-(N-methylcarbamoyl)-adenosine(Cl—IB-MECA), which is a substance having high affinity and selectivityfor the A₃ receptor compared to the adenosine A₁ and A₂ receptors. Onthe other hand, disadvantage has been noted that adenosine A₃ receptorantagonists, which exhibit high affinity and selectivity are mostlynonpurine-based bicyclic ring compounds, not nucleoside backbones, andbecause they exhibit high activity in human receptors, but they havelittle or no activity against the A₃ receptor in rats, it is notpossible to test animals that are essential for the development of drugsthat can be clinically applied (Baraldi, P G et al., Curr. Med. Chem.,12, 1319-1329, 2005). However, compared to the nonpurine-based bicyclicring compound, the nucleoside-based compound exhibits high affinity andselectivity regardless of species, so it is considered to have a greatadvantage in animal experiments, so the potential for development as anew drug is very high. Therefore, it is an urgent task to derive aselective adenosine A₃ receptor antagonist from this type.

On the other hand, the retina is a transparent and thin film located onthe innermost part of the eye ball wall and in contact with the vitreousbody in the eye ball. It converts the optical information of an objectinto an electrical signal and serves as the primary visual informationorgan that delivers images through the visual nerve to the centralvisual area of the brain. The retina consists of more than 100 millionlight-sensing cells (light-sensitive photoreceptor cells), more than 1million visual nerve cells, ganglion cells, and numerous nerve cellsthat act as a wire for connecting them, and thus it is the mostsophisticated tissue in our body. The macula lutea, the central part ofthe retina that distinguishes color and objects and represents vision,consists of a light-sensitive photoreceptor cells layer composed ofconical cells and a ganglion cells layer, which makes the retina thinand converts from the electrical signal of the image into chemicalsignal in bright light and transmits to the brain through the axon ofthe ganglion cells, the optic nerve, and the retina other than themacula lutea recognizes the periphery and plays a major role in thedark. On the other hand, if an abnormality occurs in the retina due toaging or external factors, it gradually leads to blindness with visualimpairment that causes problems with visual acuity and visual filed.

Retinal disease is occurred by abnormalities in the retina peripheraltissue and retinal detachment and It is classified into three types ofretinal detachment in which the retina is detached into the back of theeye ball to cause visual impairment; peripheral retinal degenerationcausing abnormalities in retina peripheral tissue; and maculardegeneration causing abnormalities in the macula lutea. Once the retinais separated from the pigment epithelial layer, it is unable to receiveoptical information regarding the image, and it is also unable to supplynutrients from the choroid and thus nerve cells cannot function and ifthis condition is left unattended, permanent retinal atrophy occurs,leading to blindness. The main cause of blindness is a retinal diseasewhich is mainly caused by aging and also can be caused by genetic orexcessive myopia, trauma, etc. and is the second most common ophthalmicdisease after cataracts. Retinal disease is not a fatal disease thatleads to death, but the onset has increased rapidly in recent years dueto industrialization and dietary changes along with the increase in theelderly population and thus it is necessary to develop a composition forthe treatment of retinal diseases that can be supplied in the form ofcrude drugs that have been conventionally ingested and are not atherapeutic agent synthesized artificially besides the surgical method.

DISCLOSURE Technical Problem

An object of the present invention is to provide a pharmaceuticalcomposition, an oral administration agent or eye drops which caneffectively prevent or treat retinal diseases.

Another object of the present invention is to provide a pharmaceuticalcomposition, an oral administration agent or eye drops which caneffectively prevent or treat optic nerve diseases.

Technical Solution

In order to achieve the above object, the present invention provides apharmaceutical composition for preventing or treating retinal diseasecomprising a compound represented by the following Chemical Formula 1 ora pharmaceutically acceptable salt thereof as an active ingredient.

Also, the present invention provides an oral administration agent forpreventing or treating retinal disease comprising a compound representedby the following Chemical Formula 1 or a pharmaceutically acceptablesalt thereof as an active ingredient.

In addition, the present invention provides an eye drop for preventingor treating retinal disease comprising a compound represented by thefollowing Chemical Formula 1 or a pharmaceutically acceptable saltthereof as an active ingredient.

In order to achieve the above other object, the present inventionprovides a pharmaceutical composition for preventing or treating opticnerve disease comprising a compound represented by the followingChemical Formula 1 or a pharmaceutically acceptable salt thereof as anactive ingredient.

Also, the present invention provides an oral administration agent forpreventing or treating optic nerve disease comprising a compoundrepresented by the following Chemical Formula 1 or a pharmaceuticallyacceptable salt thereof as an active ingredient.

In addition, the present invention provides an eye drop for preventingor treating optic nerve disease comprising a compound represented by thefollowing Chemical Formula 1 or a pharmaceutically acceptable saltthereof as an active ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

Advantageous Effects

The present invention relates to a composition for preventing ortreating retinal disease or optic nerve disease comprising a specificadenosine derivative as an active ingredient. The adenosine derivativecan suppress the inflammatory response by inhibiting the expression ofinflammation-related proteins, VEGF and inflammatory cytokines inphotoreceptor cells derived from the mouse retina, and can inhibitapoptosis induced by glutamic acid, and eye drops prepared by comprisingthe same have shown an effect of effectively protecting retinal ganglioncells in experiments with mice. Therefore, the present invention caneffectively prevent or treat retinal disease or optic nerve disease.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a result of confirming a change in the protein expressionrelated to angiogenesis and inflammatory response when Compound A istreated in an example of the present invention.

FIG. 2 shows a result of confirming a change in the expression of VEGFand inflammatory cytokines when Compound A is treated in the Example ofFIG. 1.

FIG. 3 shows a result of confirming a change in the expression of mRNArelated to angiogenesis and inflammatory response when Compound A istreated in the Example of FIG. 1.

FIG. 4 shows a result of confirming the cytotoxicity of thephotoreceptor cells derived from the mouse retina when Compound A istreated in another example of the present invention.

FIG. 5 shows a result of confirming the cell protective effect ofCompound A against the cell death induced by glutamic acid in theExample of FIG. 4 ((n=6), (a) pretreatment of Compound A for 30 minutes;(b) pretreatment of Compound A for 1 hour).

FIG. 6 shows a result of observing the effect of inhibiting the celldeath of Compound A in the Example of FIG. 4 by a microscope throughTUNEL assay and DAPI staining.

FIG. 7 shows a result of confirming the mitochondrial protective effectof Compound A in the Example of FIG. 4 ((a) JC-1 aggregate, (b)distribution of normal group cells, (c) distribution of experimentalgroup cells treated only with glutamic acid, (d) distribution ofexperimental group cells treated with glutamic acid after pretreatmentwith Compound A, (e) distribution of control group cells treated withCCCP only).

FIG. 8 shows a result of confirming the effect of inhibiting the caspaseactivity of Compound A in the Example of FIG. 4 ((a) activity of caspase3/7, (b) activity of caspase 8).

FIG. 9 and FIG. 10 show results of confirming the expression change ofthe protein related to cell death by Compound A in the Example of FIG. 4(FIG. 9, (a) protein expression change; (b) AIF protein expressionchange in the cell nucleus; (c) cytochrome c protein expression changein the cytoplasm; (d) mitochondrial AIF protein expression change; and(e) mitochondrial cytochrome c protein expression change; FIG. 10a , (a)protein expression change, (b) RIP protein expression change, (c) RIP3protein expression change; FIG. 10b , (d) pBcl2 protein expressionchange; (e) Bcl2 protein expression change; (f) pBad protein expressionchange; (g) Bad protein expression change; FIG. 10c , (h) BID proteinexpression changes; (i) caspase 8 protein expression changes; (j)cleaved caspase 9 protein expression changes; and (k) cleaved caspase 3protein expression changes).

FIG. 11 is a graph showing a result of measuring the number of retinalganglion cells in the eyeball of a mouse according to another example ofthe present invention.

FIG. 12 is a photograph observing histological changes of the mouseretina according to the Embodiment of FIG. 11.

BEST MODE

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition forpreventing or treating retinal disease comprising a compound representedby Chemical Formula 1 or a pharmaceutically acceptable salt thereof asan active ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy, or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

At this time, the retinal disease may be diabetic retinopathy orage-related macular disease, but it is not limited thereto.

Preferably, in the above Chemical Formula 1, A may be O or S, R may bemethyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl substitutedindependently or optionally with 1 or 2 or more substituents selectedfrom the group consisting of fluoro, chloro, bromo or C₁ to C₃ alkoxy ortoluic acid, and Y may be H or Cl.

More preferably, A is O or S, R is methyl, ethyl, 1-naphthylmethyl,benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl,2-methoxy-5-chlorobenzyl, 2-methoxybenzyl or 3-toluic acid, and Y is Hor Cl.

Preferred examples of the adenosine derivative represented by ChemicalFormula 1 according to the present invention are as follows:

-   (1) (2R,3R,4S)-2-(2-chloro-6-(3-fluorobenzylamino)-9H-purin-9-yl)    tetrahydrothiophene-3,4-diol;-   (2) (2R,3R,4S)-2-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)    tetrahydrothiophene-3,4-diol;-   (3) (2R,3R,4S)-2-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)    tetrahydrothiophene-3,4-diol;-   (4) (2R,3R,4S)-2-(2-chloro-6-(2-chlorobenzylamino)-9H-purin-9-yl)    tetrahydrothiophene-3,4-diol;-   (5)    (2R,3R,4S)-2-(2-chloro-6-(5-chloro-2-methoxybenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;-   (6)    (2R,3R,4S)-2-(2-chloro-6-(2-methoxybenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;-   (7)    (2R,3R,4S)-2-(2-chloro-6-(naphthalen-1-ylmethylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;-   (8)    3-((2-chloro-9-((2R,3R,4S)-3,4-dihydroxytetrahydrothiophen-2-yl)-9H-purin-6-ylamino)methyl)benzoic    acid;-   (9)    2-(2-chloro-6-methylamino-purin-9-yl)tetrahydrothiophene-3,4-diol;-   (10)    (2R,3R,4S)-2-(6-(3-fluorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;-   (11)    (2R,3R,4S)-2-(6-(3-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;-   (12)    (2R,3R,4S)-2-(6-(3-bromobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;    and-   (13)    (2R,3R,4R)-2-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-3,4-diol.

Most preferably, the compound represented by the Chemical Formula 1 maybe a compound represented by Chemical Formula 2:

In addition, the present invention provides an oral administration agentfor preventing or treating retinal disease comprising a compoundrepresented by Chemical Formula 1 or a pharmaceutically acceptable saltthereof as an active ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy, or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

At this time, the retinal disease may be diabetic retinopathy orage-related macular disease, but it is not limited thereto.

Preferably, in the above Chemical Formula 1, A may be O or S, R may bemethyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl substitutedindependently or optionally with 1 or 2 or more substituents selectedfrom the group consisting of fluoro, chloro, bromo or C₁ to C₃ alkoxy ortoluic acid, and Y may be H or Cl.

More preferably, A is O or S, R is methyl, ethyl, 1-naphthylmethyl,benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl,2-methoxy-5-chlorobenzyl, 2-methoxybenzyl or 3-toluic acid, and Y is Hor Cl.

Most preferably, the compound represented by the Chemical Formula 1 maybe a compound represented by Chemical Formula 2:

Furthermore, the present invention provides An eye drop for preventingor treating retinal disease comprising a compound represented byChemical Formula 1 or a pharmaceutically acceptable salt thereof as anactive ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy, or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

In this case, the retinal disease may be diabetic retinopathy orage-related macular disease, but it is not limited thereto.

Preferably, in the above Chemical Formula 1, A may be O or S, R may bemethyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl substitutedindependently or optionally with 1 or 2 or more substituents selectedfrom the group consisting of fluoro, chloro, bromo or C₁ to C₃ alkoxy ortoluic acid, and Y may be H or Cl.

More preferably, A is O or S, R is methyl, ethyl, 1-naphthylmethyl,benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl,2-methoxy-5-chlorobenzyl, 2-methoxybenzyl or 3-toluic acid, and Y is Hor Cl.

Most preferably, the compound represented by the Chemical Formula 1 maybe a compound represented by Chemical Formula 2:

The present invention provides a pharmaceutical composition forpreventing or treating optic nerve disease comprising a compoundrepresented by Chemical Formula 1 or a pharmaceutically acceptable saltthereof as an active ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy, or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

At this time, the retinal disease may be diabetic retinopathy orage-related macular disease, but it is not limited thereto.

Preferably, in the above Chemical Formula 1, A may be O or S, R may bemethyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl substitutedindependently or optionally with 1 or 2 or more substituents selectedfrom the group consisting of fluoro, chloro, bromo or C₁ to C₃ alkoxy ortoluic acid, and Y may be H or Cl.

More preferably, A is O or S, R is methyl, ethyl, 1-naphthylmethyl,benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl,2-methoxy-5-chlorobenzyl, 2-methoxybenzyl or 3-toluic acid, and Y is Hor Cl.

Most preferably, the compound represented by the Chemical Formula 1 maybe a compound represented by Chemical Formula 2:

The adenosine derivative represented by the above Chemical Formula 1according to the present invention can be used in the form of apharmaceutically acceptable salt. As the salt, acid addition saltsformed by various pharmaceutically acceptable organic or inorganic acidsare useful. Suitable organic acids include, for example, carboxylicacid, phosphonic acid, sulfonic acid, acetic acid, propionic acid,octanoic acid, decanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, adipic acid, malic acid, tartaric acid, citric acid,glutamic acid, aspartic acid, maleic acid, benzoic acid, salicylic acid,phthalic acid, phenylacetic acid, benzenesulfonic acid,2-naphthalenesulfonic acid, methylsulfuric acid, ethylsulfuric acid,dodecylsulfuric acid, etc. Suitable inorganic acids include, forexample, halogen acids such as hydrochloric acid and sulfuric acid orphosphoric acid and the like.

The adenosine derivative represented by the above Chemical Formula 1according to the present invention may include all salts, hydrates andsolvates that can be prepared by conventional methods, as well aspharmaceutically acceptable salts.

In addition, the pharmaceutical composition for preventing or treatingretinal disease or optic nerve disease according to the presentinvention may include a pharmaceutically acceptable carrier, excipientor diluent in addition to the above-mentioned active ingredients foradministration. The carrier, excipients and diluents include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia rubber, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methyl cellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can beformulated and used in the form of oral formulations such as powders,granules, tablets, capsules, suspensions, emulsions, syrups, aerosols,etc., external preparations, suppositories or sterile injectablesolutions according to a conventional method. In detail, whenformulated, it may be prepared using diluents or excipients such asfillers, weighting agents, binders, wetting agents, disintegratingagents, surfactants, etc., which are commonly used. Solid formulationsfor oral administration include tablets, pills, powders, granules,capsules, etc., but it is not limited thereto. Such a solid preparationmay be prepared by mixing at least one excipient such as starch, calciumcarbonate, sucrose, lactose, gelatin and the like in addition to acompound represented by the above Chemical Formula 1 or apharmaceutically acceptable salt thereof. Furthermore, in addition tosimple excipients, lubricants such as magnesium stearate and talc arealso used. Examples of the liquid formulations for oral administrationinclude suspensions, solutions, emulsions, syrups and the like, andvarious excipients such as wetting agents, sweeteners, fragrances,preservatives and the like may be included in addition to water andliquid paraffin. Formulations for parenteral administration includesterile aqueous solutions, non-aqueous solutions, suspensions,emulsions, freeze-dried preparations and suppositories. Examples of thenon-aqueous solution and the suspension include propylene glycol,polyethylene glycol, vegetable oil such as olive oil, injectable estersuch as ethyl oleate, and the like. As the base of the suppository,witepsol, macrogol, tween 61, cacao butter, laurinum, glycerogelatin andthe like can be used.

The suitable dosage of the composition of the present invention dependson the patient's condition and body weight, the degree of disease, drugform and time, but it can be appropriately selected by those skilled inthe art.

In addition, the present invention provides an oral administration agentfor preventing or treating optic nerve disease comprising a compoundrepresented by Chemical Formula 1 or a pharmaceutically acceptable saltthereof as an active ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy, or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

In this case, the optic nerve disease may be selected from the groupconsisting of ischemic optic neuropathy, traumatic optic neuropathy andcompressive optic neuropathy, but it is not limited thereto.

Preferably, in the above Chemical Formula 1, A may be O or S, R may bemethyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl substitutedindependently or optionally with 1 or 2 or more substituents selectedfrom the group consisting of fluoro, chloro, bromo or C₁ to C₃ alkoxy ortoluic acid, and Y may be H or Cl.

More preferably, A is O or S, R is methyl, ethyl, 1-naphthylmethyl,benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl,2-methoxy-5-chlorobenzyl, 2-methoxybenzyl or 3-toluic acid, and Y is Hor Cl.

Most preferably, the compound represented by the Chemical Formula 1 maybe a compound represented by Chemical Formula 2:

The oral administration agent for preventing or treating retinal diseaseor optic nerve disease may be formulated as a solid preparation or aliquid preparation of a compound represented by the above ChemicalFormula 1 and/or a pharmaceutically acceptable salt thereof.

The solid preparation may be tablets, pills, powders, granules,capsules, etc. and the liquid preparation may be suspensions, solutions,emulsions, syrups, etc., but they are not limited thereto.

The oral administration agent for preventing or treating retinal diseaseor optic nerve disease may further include an excipient, that is atleast one selected from the group consisting of methyl cellulose (MC),sucrose, lactose, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG),magnesium stearate, calcium carbonate, gelatin, talc, distilled water(DW), liquid paraffin, etc., preferably at least one selected from thegroup consisting of methyl cellulose (MC), dimethyl sulfoxide (DMSO),polyethylene glycol (PEG) and distilled water, and more preferably, 0.5wt % of methyl cellulose.

In the oral administration agent for preventing or treating the retinaldisease or optic nerve disease according to an example of the presentinvention, the compound represented by the above Chemical Formula 1 or apharmaceutically acceptable salt thereof may be filled in a capsule as apowder form or a solution form dissolved in the above-describedexcipient, but is not limited thereto.

In addition, the present invention provides an eye drop for preventingor treating optic nerve disease comprising a compound represented byChemical Formula 1 or a pharmaceutically acceptable salt thereof as anactive ingredient:

in Chemical Formula 1, A is O or S;

R is a) straight or branched C₁ to C₅ alkyl unsubstituted, orindependently or optionally substituted with 1 or 2 or more C₆ to C₁₀aryl, b) benzyl unsubstituted, or independently or optionallysubstituted with 1 or 2 or more fluoro, chloro, bromo or straight orbranched C₁ to C₄ alkoxy, or c) benzyl substituted with hydroxycarbonyl;and Y is H or a halogen element.

In this case, the optic nerve disease may be selected from the groupconsisting of ischemic optic neuropathy, traumatic optic neuropathy andcompressive optic neuropathy, but it is not limited thereto.

Preferably, in the above Chemical Formula 1, A may be O or S, R may bemethyl, ethyl, propyl, naphthylmethyl, benzyl, benzyl substitutedindependently or optionally with 1 or 2 or more substituents selectedfrom the group consisting of fluoro, chloro, bromo or C₁ to C₃ alkoxy ortoluic acid, and Y may be H or Cl.

More preferably, A is O or S, R is methyl, ethyl, 1-naphthylmethyl,benzyl, 2-chlorobenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-bromobenzyl,2-methoxy-5-chlorobenzyl, 2-methoxybenzyl or 3-toluic acid, and Y is Hor Cl.

Most preferably, the compound represented by the Chemical Formula 1 maybe a compound represented by Chemical Formula 2:

The eye drop for preventing or treating retinal disease or optic nervedisease may include the compound represented by the above ChemicalFormula 1 and/or a pharmaceutically acceptable salt and eye dropthereof. The eye drop may include one or more selected from the groupconsisting of a solubilizer, a viscosity enhancer, an antioxidant, apreservative and a buffer solution.

In one example of the present invention, the eye drop may be a buffersolution of pH 6.8 in which Cremophor EL, glycerin, citric acid andmethylparaben are dissolved or mixed, but it is not limited thereto.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, examples of the present invention will be described indetail to understand the present invention. The present invention may,however, be embodied in many different forms and should not be limitedto the embodiments set forth herein in order to clearly illustrate thepresent invention for those skilled in the art to which the presentinvention pertains.

<Preparation Example 1> Synthesis of Adenosine Derivative Compound andPreparation of Eye Drop

Adenosine derivatives were synthesized according to the method disclosedin Korean Patent No. 10-1396092. The synthesized adenosine derivative isas follows:

(2R,3R,4S)-2-(2-chloro-6-(3-fluorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(2-chloro-6-(3-bromobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(2-chloro-6-(2-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(2-chloro-6-(5-chloro-2-methoxybenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(2-chloro-6-(2-methoxybenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(2-chloro-6-(naphthalen-1-ylmethylbenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,3-((2-chloro-9-((2R,3S,4R)-3,4-dihydroxytetrahydrothiophen-2-yl)-9H-purin-6-ylamino)methyl)benzoicacid,2-(2-chloro-6-methylamino-purin-9-yl)(2R,3S,4R)-tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(6-(3-fluorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(6-(3-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol,(2R,3R,4S)-2-(6-(3-bromobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-dioland (2R,3R,4R)-2-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)tetrahydrofuran-3,4-diol.

In subsequent experiments,(2R,3R,4S)-2-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol(hereinafter, referred to as “Compound A”) was used and for animalexperiments, the Compound A was mixed with a buffer solution of pH 6.8in which cremophor EL, glycerin, citric acid and methylparaben weredissolved, and eye drops were prepared to be included at concentrationsof 250 μM and 500 μM and 750 μM of the compound, respectively.

<Example 1> Confirmation of Inhibitory Effect of Adenosine DerivativeCompound on Angiogenesis and Inflammatory Response

1. Experimental Method

(1) Preparation and Treatment of Test Materials

Compound A to be used as a test material was prepared by dissolving inDMSO. The prepared solution was stored at −20° C. until use. Treatmentof the test material was treated after diluting to 1:1000 (v/v) in aserum-free cell culture medium. The control group was treated afterdiluting to 1:1000 (v/v) in DMSO.

(2) Analysis of Protein Expression Level in Cells when Treating TestMaterial

In order to confirm the efficacy of the test material in reactionsrelated to angiogenesis and inflammatory response of photoreceptor cellsderived from mouse retina, cells cultured for 24 hours under variousconditions were recovered and intracellular proteins were extractedusing a PRO-PREP protein extraction kit. Quantification of the extractedprotein was measured using a BCA protein assay kit. 40 μg of protein wasseparated by 10% SDS-PAGE and then attached to the PVDF membrane. Theprotein-attached membrane was reacted with TBST (Tris-buffered salinewith 0.1% Tween-20) containing 5% skim milk for 30 minutes and theprimary antibody and secondary antibody were reacted in sequence. Theprotein expression level was confirmed by photographing using a FusionFximage acquisition system. The protein expression level was analyzed byrelative values using ImageJ. Meanwhile, the antibodies and experimentalconditions used for immunoblotting are summarized in Table 1 below.

TABLE 1 Primary antibody Secondary antibody Antibody Company DilutionAntibody Company Company Dilution VEGF Cell Signaling 1:1,000 Goat-antirabbit Santacruz 1:5,000 FLT1 Santacruz 1:500  Goat-anti rabbitSantacruz 1:5,000 (VEGFR1) FLK1 Santacruz 1:500  Goat-anti rabbitSantacruz 1:5,000 (VEGFR2) Angiopoietin1 Santacruz 1:500  Rabbit-antigoat Santacruz 1:5,000 Angiopoietin2 Santacruz 1:500  Rabbit-anti goatSantacruz 1:5,000 COX2 Santacruz 1:500  Rabbit-anti goat Santacruz1:5,000 MMP2 Cell Signaling 1:1,000 Goat-anti rabbit Santacruz 1:5,000MMP9 Cell Signaling 1:1,000 Goat-anti rabbit Santacruz 1:5,000 ICAM1Cell Signaling 1:1,000 Goat-anti rabbit Santacruz 1:5,000 VCAM1 CellSignaling 1:1,000 Goat-anti rabbit Santacruz 1:5,000 β-actin Santacruz1:3,000 Rabbit-anti mouse Santacruz 1:5,000

(3) Quantitative Analysis of Inflammatory Cytokines in Cells whenTreating Test Materials

In order to confirm the efficacy of the test material in reactionsrelated to angiogenesis and inflammatory response of photoreceptor cellsderived from mouse retina, cells cultured for 24 hours under variousconditions were recovered and intracellular proteins were extractedusing a PRO-PREP protein extraction kit. Quantification of the extractedprotein was measured using a BCA protein assay kit. Quantitativeanalysis of inflammatory cytokines (TNFα, IL-1β, IL-6) present in thecells was analyzed with each ELISA kit.

(4) Quantitative Analysis of VEGF in Medium when Treating Test Materials

In order to confirm the efficacy of the test material in reactionsrelated to angiogenesis of photoreceptor cells derived from mouseretina, the medium of cells cultured for 24 hours under variousconditions was collected after centrifugation.

Quantitative analysis of VEGF present in the medium was analyzed withthe Mouse VEGF Quantikine ELISA Kit.

(5) Analysis of mRNA Expression Level in Cells when Treating TestMaterials

In order to confirm the efficacy of the test material in reactionsrelated to angiogenesis and inflammatory response of photoreceptor cellsderived from mouse retina, cells cultured for 6 hours under variousconditions were treated with TRIzol solution to isolate total RNA incells. After the cDNA was synthesized using specific primers for thetarget gene (Table 2) and reverse transcriptase in the isolated totalRNA, expression of the related factors was confirmed. mRNA expressionlevels were analyzed by expressing as relative values using ImageJsoftware.

At this time, primers and experimental conditions used for PCR analysisare summarized in Table 2 below.

TABLE 2 Annealing Gene Gene ID Primer Temp. Cycle Length MMP2NM_008610.3 GCTGCGCTTTTCTCGAATCC 60° C. 30 375 GTAAACAAGGCTTCATGGGGGMMP9 NM_013599.4 CGCTCATGTACCCGCTGTAT 65° C. 30 345 TGTCTGCCGGACTCAAAGACVEGF NM_001025250.3 CTCCGTAGTAGCCGTGGTCT 65° C. 30 496GCTTCGCTGGTAGACATCCA FLT1 NM_010228.3 TCTAGAAGACTCGGGCACCT 65° C. 30 403(VEGFR1) CGTGATCAGCTCCAGGTTTG FLK1 X70842.1 AACACGTGGACTCTGTCCTCC 65° C.30 323 (VEGFR2) GAAGAGCACGCAAACCTTCC TNFα NM_013693.3GACAAGCCTGTAGCCCACG 60° C. 30 482 TGGGGGCTGGGTAGAGAATG IL-6 NM_031168.2GCCTTCTTGGGACTGATGCT 65° C. 30 475 TGGAAATTGGGGTAGGAAGGAC COX2 N/AGTATCAGAACCGCATTGCCTC 60° C. 30 526 CGGCTTCCAGTATTGAGGAGAACAGAT ICAM1N/A CCTGTTTCCTGCCTCTGAAG 60° C. 30 528 GTCTGCTGAGACCCCTCTTG VCAM1 N/ATCTAGAAGACTCGGGCACCT 60° C. 30 403 CGTGATCAGCTCCAGGTTTG GAPDHNM_001289726.1 GTGCCGTTGAATTTGCCGTGA 60° C. 30 325 ATGGTGAAGGTCGGTGTGAAC

(6) Statistical Analysis

Measured values of each experimental group were analyzed statisticallythrough Microsoft's Excel (2007). The outliers of the result values ofeach experimental group were determined by obtaining the quartiles, andthe analysis of variance by one-way design of experiment of each datawas performed and the results were expressed as the mean and standarddeviation. In addition, the effectiveness was analyzed at a significancelevel of 0.05 or less through F-test and t-test (equal variance andheteroscedasticity).

2. Experimental Results

(1) Changes in Expression of Proteins Related to Angiogenesis andInflammatory Response

In relation to high mobility group box 1 (HMGB-1), it has been reportedthat the expression of HMGB-1 was increased when pressure was applied tophotoreceptor cells derived from the mouse retina, and when HMGB-1 wastreated to photoreceptor cells, angiogenesis-related factors andinflammatory responses increase (Bohm M R et al., Lab Invest, 96,409-427, 2016). Accordingly, in the present invention, the efficacy ofadenosine derivatives against angiogenic response and inflammatoryresponse according to damage to photoreceptor cells was verified basedon the above reference.

After treatment with adenosine derivatives Compound A and HMGB-1, theresults of analyzing protein expression changes of angiogenic responsefactors such as VEGF, VEGF receptor 1, VEGF receptor 2, angiopoietin1and angiopoietin2 enzymes and inflammatory response factors such asCOX2, MMP2, MMP9, ICAM1 and VCAM1 enzymes were shown in FIG. 1a and FIG.1 b.

That is, FIG. 1a and FIG. 1a show that the expression of VEGF, VEGFR1,VEGFR2 and ANG2, which are angiogenesis-related factors, issignificantly increased by HMGB-1 (5 μg/mL), and it is shown thatprotein expression of angiogenesis-related factors increased in aconcentration-dependent manner is decreased by treatment with CompoundA. The expression of VEGF and VEGFR2, COX2 and MMP2 proteins wassignificantly reduced at concentrations of at least 0.5 μM of Compound Aand expression of VEGFR1, ANG2 and MMP9 was significantly decreased atconcentrations of at least 0.1 μM. ANG1 protein expression wassignificantly increased at 2 μM concentration and ICAM1 and VCAM1protein expression was significantly decreased at 2 μM concentration.

(2) Change in Expression of VEGF and Inflammatory Cytokines

As a result of performing a quantitative analysis through the ELISAanalysis method to confirm the efficacy of Compound A inangiogenesis-related response and inflammatory response of mouse retinalphotoreceptor cells by HMGB-1, as shown in FIG. 2, the amount of VEGFincreased by HMGB-1 was significantly decreased by Compound A of 0.1 μMor more. The amount of inflammatory cytokines (TNFα, IL-1β and IL-6) incells was increased by HMGB-1, but TNFα was significantly reduced bytreatment with Compound A at concentrations of 0.1 μM or higher andIL-1β and IL-6 was significantly reduced by treatment with Compound A atconcentrations of 1 μM or higher.

(3) Changes in mRNA Expression Related to Angiogenesis and InflammatoryResponse

As a result of analyzing mRNA expression changes of VEGF, VEGF receptor1, VEGF receptor 2 enzymes, which are angiogenesis-related factors, andTNFα, IL-6, COX2, MMP2, MMP9, ICAM1 and VCAM1 enzymes, which are factorsrelated to inflammatory response, after treatment with compound A, asshown in FIG. 3a and FIG. 3b , mRNA expression of VEGF, VEGFR1 andVEGFR2, which are angiogenesis-related factors, was significantlyincreased by HMGB-1. However, the mRNA expression of theangiogenesis-related factors increased by treatment with Compound A wasdecreased in a concentration-dependent manner. The expression of VEGFand VEGFR1 showed a significant decrease when Compound A was treated ata concentration of 0.1 μM or more, and the expression of VEGFR2 wassignificantly decreased at concentrations of 0.5 μM or more. Inaddition, the mRNA expression of TNFα, IL-6, COX2, MMP2, MMP9, ICAM1 andVCAM1, which are factors related to the inflammatory response, was alsosignificantly increased by HMGB-1 in the control group but decreased ina concentration-dependent manner by treatment with Compound A. Theexpression of COX2 and IL-6 was significantly inhibited by treatmentwith Compound A of 0.1 μM or more and the expression of TNFα and MMP9was significantly reduced at concentrations of 0.5 μM or more. Theexpression of MMP2, ICAM1 and VCAM1 was significantly reduced bytreatment with Compound A at 1 μM or more.

<Example 2> Protective Effect of Adenosine Derivatives on RetinalPhotoreceptor Cell

1. Experimental Method

(1) Preparation and Treatment of Test Materials

Compound A to be used as a test material was prepared by dissolving inDMSO. The prepared solution was stored at −20° C. until use. Inaddition, 10 mg of CCCP was dissolved in 1 mL of DMSO to prepare a 50 mMCCCP solution. The prepared solution was stored at −20° C. until use.Treatment of the test material was performed after diluting with aserum-free cell culture medium in 1:1000 (v/v). The control group wastreated after diluting with DMSO in 1:1000 v/v).

(2) TUNEL Analysis

Quantitative analysis of apoptosis cells was analyzed by DeadEnd™Fluorometric TUNEL System. Cells were pretreated with 1 μM of Compound Afor 1 hour and then cultured for 8 hours in a medium containing 5 mMglutamic acid. Cells were washed twice with PBS and fixed with 10%formalin for 15 minutes. Fixed cells were exposed to 0.1% Triton X-100for 10 minutes. After the cells were reacted with dUTP containingfluorescence, the cut DNA portion was stained and then fixed on a slidewith a mounting solution containing DAPI. The stained cells wereobserved through a fluorescence microscope.

(3) Caspase 3/7 Activity Assay

The activity of intracellular caspase 3/7 was analyzed by Caspase-Glo3/7 assay system. Cells were pretreated with 1 μM of Compound A for 1hour and then cultured for 24 hours in a medium containing 5 mM glutamicacid. After adding the kit solution in the same amount as the cellculture medium and reacting for 1 hour, the relative activity wasanalyzed by measuring the amount of generated luminescence through amicroplate reader.

(4) Caspase 8 Activity Assay

The activity of intracellular caspase 8 was analyzed by Caspase-Glo 8assay system. Cells were pretreated with 1 μM of Compound A for 1 hourand then cultured for 24 hours in a medium containing 5 mM glutamicacid. After adding the kit solution in the same amount as the cellculture medium and reacting for 1 hour, the relative activity wasanalyzed by measuring the amount of generated luminescence through amicroplate reader.

(5) Mitochondrial Cell Membrane Potential Analysis

Intracellular mitochondrial cell membrane potential was analyzed usingJC-1 reagent. Cells were pretreated with 1 μM of Compound A for 1 hourand then cultured for 6 hours in a medium containing 5 mM glutamic acid.Thereafter, 5 mM of JC-1 was treated and incubated for 30 minutes. JC-1aggregates and JC-1 monomers were analyzed by using an Attune AcousticFocusing Cytometer at excitation wavelengths of 485±11 nm and 535±17.5nm and emission wavelengths of 530±15 and 590±17.5 nm.

(6) Intercellular Protein Expression Level Analysis

In order to confirm the efficacy of a test material forangiogenesis-related response and inflammatory response of a mouseretina-derived photoreceptor cell, cells cultured for 24 hours undervarious conditions were collected and intracellular protein wasextracted by using a PRO-PREP protein extraction kit. Quantification ofthe extracted protein was measured using a BCA protein assay kit. 40 μgof protein was separated by 10% SDS-PAGE and then attached to a PVDFmembrane. The protein-attached membrane was reacted with Tris-bufferedsaline with 0.1% Tween-20 (TBST) containing 5% skim milk for 30 minutes,followed by reactions of primary and secondary antibodies in sequence.The protein expression level was confirmed using photograph by a FusionFximage acquisition system. The protein expression level was analyzed byexpressing as a relative value using ImageJ. Meanwhile, the antibodiesand experimental conditions used in the immunoblot analysis aresummarized in Table 3 below.

TABLE 3 Primary antibody Antibody Company Dilution Mouse anti-AIFSantacruz 1:1,000 Mouse anti-cytochrome c Santacruz 1:1,000 Mouseanti-LaminB Santacruz 1:1,000 Mouse anti-COX IV Santacruz 1:1,000 Mouseanti-β-actin Santacruz 1:3,000 Mouse anti-GAPDH Santacruz 1:3,000 Mouseanti-Bcl₂ Santacruz 1:500  Mouse anti-pBcl₂ Santacruz 1:500  Mouse:anti-BID Santacruz 1:200  Rabbit anti-BAD Cell signaling 1:1,000 Rabbitanti-pBAD Cell signaling 1:1,000 Rabbit anti-cleaved caspase 3 Cellsignaling 1:1,000 Mouse anti-caspase 8 Cell signaling 1:1,000 Rabbitanti-cleaved caspase 9 Cell signaling 1:1,000 Rabbit anti-RIP Santacruz1:500  Mouse anti-RIP3 Santacruz 1:500 

(7) Statistical Analysis

The measured value of each experimental group was statistically analyzedthrough SPSS 23.0 of IBM. When the equal variance of each experimentalgroup was one variance, the analysis was performed by one-way analysisof variance and Tukeys test and in the case of heteroscedasticity, itwas performed by one-way analysis of variance and Welchs t-test. Theexperimental group showing significant statistical difference from thenormal group was shown as *P<0.05, *P<0.01, ***P<0.001, and theexperimental group showing significant statistical difference from thecontrol group treated with only glutamate was shown as #P<0.05, P<0.01,#P<0.001.

2. Experimental Results

(1) Cytotoxicity of Glutamic Acid

After diluting glutamic acid in the cultured cells in the medium andtreating the cells with concentrations of 0-9 mM, the cells wasincubated for 24 hours, treated with a reagent for cell proliferationanalysis (CellTiter96ter AQueous One Solution Cell Proliferation AssayKit) and after 1 hour, the cell viability was measured to compare andanalyze the cytotoxicity of glutamic acid to photoreceptor cells derivedfrom the mouse retina. As a result, as shown in FIG. 4, it was found toinduce a significant cell viability decrease in aconcentration-dependent manner at concentrations of 3 mM or more. TheIC₅₀ value of glutamic acid was confirmed to be 5.1±0.5 mM.

(2) Cell Protective Effect of Adenosine Derivatives

As a result of confirming the cell protective effect of Compound A fromapoptosis induced by glutamic acid, as shown in FIG. 5 (n=6), it wasconfirmed that the pretreatment of Compound A inhibits cell viabilityinhibition by glutamic acid in a concentration-dependent manner up to aconcentration of 1 μM. This experimental result means that Compound Ahas a protective effect against glutamic acid-induced apoptosis. TheEC₅₀ (50% effective concentration) levels of Compound A were 0.31±0.08μM and 0.35±0.06 μM for pretreatment for 30 min and 1 hour,respectively, which were similar to each other.

(3) Inhibitory Effect of Adenosine Derivatives on Apoptosis

In order to confirm the effect of inducing apoptosis by glutamic acidand inhibiting the effect of Compound A, cells undergoing apoptosis wereobserved under a microscope through TUNEL assay and DAPI staining. As aresult, as shown in FIG. 6, it was confirmed that TUNEL-positivereactions were observed in a large number of nuclei in cells treatedwith glutamic acid only, but the number was significantly reduced incells pretreated with Compound A. These results indicate that theCompound A inhibits apoptosis by glutamic acid.

(4) Mitochondrial Protective Effect of Adenosine Derivatives

Apoptosis by glutamic acid has been reported to be associated withdamage to mitochondria. Thus, to confirm what effect Compound A has onmitochondrial damage to glutamic acid, the mitochondrial membranepotential difference was analyzed through a flow cytometer. As a result,as shown in FIG. 7 and Table 4 below, it was confirmed that JC-1aggregates were decreased in cells treated with glutamic acid only.Similar cell distribution could be confirmed by treatment of CCCPinducing mitochondrial damage. However, in the experimental grouppretreated with Compound A, the distribution of cells was found to besimilar to that of the normal group. These results indicate thatCompound A inhibits the damage of intercellular mitochondria by glutamicacid.

TABLE 4 Control Glutamate Glutamate + Compound A CCCP R1 86.22 ± 1.1236.13 ± 10.42** 71.70 ± 1.60***^(,#)  9.42 ± 1.48*** R2 13.78 ± 1.1263.87 ± 10.42** 28.30 ± 1.60***^(,#) 90.58 ± 1.48***

(5) Confirmation of Caspase Activity

Caspase is known to regulate cell death. Thus, as a result of confirmingthe action of caspase in cell damage by glutamic acid and the cellprotective effect of compound A, as shown in FIG. 8, the activities ofcaspase 3/7 of cells (FIG. 8(a)) and caspase 8 of cells (FIG. 8(b))treated with glutamic acid only were significantly increased, and as aresult of pretreatment of Compound A, the activity of caspase 3/7 andcaspase 8 was inhibited in a concentration-dependent manner. Theseresults indicate that Compound A inhibits cell death by activation ofcaspase.

(6) Changes in Expression of Apoptosis-Related Proteins

In order to confirm the efficacy of Compound A on cell death by glutamicacid, changes in AIF and cytochrome c, which are proteins related to theapoptosis, were analyzed. As a result, as shown in FIG. 9, it wasconfirmed that in cells treated with glutamic acid only, it wasconfirmed that AIF and cytochrome c, present in the mitochondria,migrated to the nucleus and cytoplasm, respectively and as a result ofpretreatment of Compound A, the expression of AIF and cytochrome c inthe nucleus and cytoplasm decreased significantly. Namely, it isconfirmed that Compound A significantly inhibits the protein transportchange. These results indicate that Compound A inhibits damage to cellsby inhibiting damage to mitochondria.

In addition, in order to confirm the mechanism of apoptosis by glutamicacid and the effect of Compound A on it, as a result of analyzing thechanges of the apoptosis-related protein, as shown in FIG. 10a to FIG.10c , it was confirmed that the expression of RIP related to apoptosiswas significantly decreased by glutamic acid, but in the experimentalgroup pretreated with Compound A, it was increased again in aconcentration-dependent manner. In addition, although the expression ofpBcl2, Bcl2, pBad, Bad and BID proteins related to mitochondrial damagewas significantly changed by glutamic acid, it was confirmed that theexperimental group pretreated with Compound A recovered to the sameexpression level as the normal group. It was confirmed that caspase 8,cleaved caspase 9 and cleaved caspase 3 were also significantlyincreased by glutamic acid, but they were significantly decreased in theexperimental group pretreated with Compound A. These results indicatethat Compound A protects cells by inhibiting apoptosis by glutamic acid.

<Example 3> Experiment of Protective Effect of Eye Drop ContainingAdenosine Derivatives on Optic Nerve

The following in vivo animal experiments were performed to examine theprotective efficacy of the eye drop of the derivatives of the presentinvention for the optic nerve. The eye drop prepared in the PreparationExample 1 was administered into the eyes of 3-month-old normal DAB 2Jmice. As a positive control, xalatan, an eye drop used as a therapeuticagent for glaucoma, was administered in the same way, and as a negativecontrol, no treatment was given to the experimental animals.

Subsequently, the number of retinal ganglion cells (RGCs) of theexperimental animals after 4 months was measured, and the cross-sectionswere observed, and the results were shown in FIG. 11 and FIG. 12,respectively. In FIG. 11 and FIG. 12, Control represents a negativecontrol, Drug 250, 500 and 750 are eye drops containing the derivativecompound (Compound A) at concentrations of 250 μM, 500 μM and 750 μM,respectively, and Xalatan represents a positive control.

Referring to FIG. 11 and FIG. 12, it was confirmed that there was aneffect of protecting the optic nerve depending on the dose of the eyedrop in mice administered with the eye drop containing the adenosinederivative of the present invention.

While the present invention has been particularly described withreference to specific embodiments thereof, it is apparent that thisspecific description is only a preferred embodiment and that the scopeof the present invention is not limited thereby to those skilled in theart. That is, the practical scope of the present invention is defined bythe appended claims and their equivalents.

1. A pharmaceutical composition for preventing or treating retinaldisease comprising a compound represented by Chemical Formula 1 or apharmaceutically acceptable salt thereof as an active ingredient:

in Chemical Formula 1, A is O or S; R is a) straight or branched C₁ toC₅ alkyl unsubstituted, or independently or optionally substituted with1 or 2 or more C₆ to C₁₀ aryl, b) benzyl unsubstituted, or independentlyor optionally substituted with 1 or 2 or more fluoro, chloro, bromo orstraight or branched C₁ to C₄ alkoxy or c) benzyl substituted withhydroxycarbonyl; and Y is H or a halogen element. 2-22. (canceled) 23.The pharmaceutical composition for preventing or treating retinaldisease of claim 1, wherein the retinal disease is diabetic retinopathyor age-related macular disease.
 24. The pharmaceutical composition forpreventing or treating retinal disease of claim 1, wherein the compoundrepresented by the Chemical Formula 1 is a compound represented byChemical Formula 2:


25. The pharmaceutical composition for preventing or treating retinaldisease of claim 1, wherein the pharmaceutical composition is an oraladministration agent.
 26. The pharmaceutical composition for preventingor treating retinal disease of claim 25, further comprising at least oneexcipient selected from the group consisting of methyl cellulose (MC),dimethyl sulfoxide (DMSO), polyethylene glycol (PEG) and distilledwater.
 27. The pharmaceutical composition for preventing or treatingretinal disease of claim 26, wherein the excipient comprises 0.5 wt % ofmethyl cellulose.
 28. The pharmaceutical composition for preventing ortreating retinal disease of claim 25, wherein the compound representedby the Chemical Formula 1 or a pharmaceutically acceptable salt thereofis filled in a capsule in powder form.
 29. The pharmaceuticalcomposition for preventing or treating retinal disease of claim 25,wherein the compound represented by the Chemical Formula 1 is a compoundrepresented by Chemical Formula 2:


30. The pharmaceutical composition for preventing or treating retinaldisease of claim 1, wherein the pharmaceutical composition is an eyedrop.
 31. The pharmaceutical composition for preventing or treatingretinal disease of claim 30, wherein the compound represented by theChemical Formula 1 is a compound represented by Chemical Formula 2:


32. A pharmaceutical composition for preventing or treating optic nervedisease comprising a compound represented by Chemical Formula 1 or apharmaceutically acceptable salt thereof as an active ingredient:

in Chemical Formula 1, A is O or S; R is a) straight or branched C₁ toC₅ alkyl unsubstituted, or independently or optionally substituted with1 or 2 or more C₆ to C₁₀ aryl, b) benzyl unsubstituted, or independentlyor optionally substituted with 1 or 2 or more fluoro, chloro, bromo orstraight or branched C₁ to C₄ alkoxy or c) benzyl substituted withhydroxycarbonyl; and Y is H or a halogen element.
 33. The pharmaceuticalcomposition for preventing or treating optic nerve disease of claim 32,wherein the optic nerve disease is selected from the group consisting ofischemic optic neuropathy, traumatic optic neuropathy, and compressiveoptic neuropathy.
 34. The pharmaceutical composition for preventing ortreating optic nerve disease of claim 32, wherein the compoundrepresented by the Chemical Formula 1 is a compound represented byChemical Formula 2:


35. The pharmaceutical composition for preventing or treating opticnerve disease of claim 32, the pharmaceutical composition is an oraladministration agent.
 36. The pharmaceutical composition for preventingor treating optic nerve disease of claim 35, further comprising at leastone excipient selected from the group consisting of methyl cellulose(MC), dimethyl sulfoxide (DMSO), polyethylene glycol (PEG) and distilledwater.
 37. The pharmaceutical composition for preventing or treatingoptic nerve disease of claim 36, wherein the excipient comprises 0.5 wt% of methyl cellulose.
 38. The pharmaceutical composition for preventingor treating optic nerve disease of claim 35, wherein the compoundrepresented by the Chemical Formula 1 or a pharmaceutically acceptablesalt thereof is filled in a capsule in powder form.
 39. Thepharmaceutical composition for preventing or treating optic nervedisease of claim 35, wherein the compound represented by the ChemicalFormula 1 is a compound represented by Chemical Formula 2:


40. The pharmaceutical composition for preventing or treating opticnerve disease of claim 32, the pharmaceutical composition is an eyedrop.
 41. The pharmaceutical composition for preventing or treatingoptic nerve disease of claim 40, wherein the optic nerve disease isselected from the group consisting of ischemic optic neuropathy,traumatic optic neuropathy, and compressive optic neuropathy.